Fibrinogen inhibits sonic hedgehog signaling and impairs neonatal cerebellar development after blood–brain barrier disruption

Significance Central nervous system (CNS) hemorrhage, the most common brain injury in premature infants, significantly increases the risk of permanent neurological impairment. The developing cerebellum is particularly vulnerable to injury from CNS bleeds, for which no specific treatment exists. In this study, we found that fibrinogen, a clotting protein that crosses a damaged blood–brain barrier, inhibits sonic hedgehog signaling in cerebellar neuron progenitors to impede the formation of new neurons in the neonatal cerebellum. Remarkably, fibrinogen depletion attenuated neuroinflammation, enhanced neurogenesis, and improved cerebellar growth in models of preterm cerebellar injury. Thus, fibrinogen-targeted therapies may counteract the harmful effects of blood in the developing CNS and provide a specific treatment to improve neurodevelopmental outcomes in preterm infants with CNS hemorrhage.


Figures S1 to S4
Tables S1 to S3 SI References

Supporting Information
Material and Methods Human subjects and MRI.Premature newborns <32 weeks' gestation at birth admitted to the intensive care nursery at the University of California, San Francisco (UCSF) between August 2011 and August 2015 were prospectively evaluated and included in this analysis.Exclusion criteria consisted of instability for MRI, congenital infection and/or clinical evidence of malformation or a genetic syndrome.Study participants received a 3-Tesla MRI soon after birth and/or at termequivalent gestational age or prior to hospital discharge with acquisition parameters as previously described (1,2).The presence and severity of cerebellar hemorrhage (CBH), white matter injury (3), and intraventricular hemorrhage (4) on MRI were contemporaneously evaluated by a pediatric neuroradiologist (AJB) blinded to clinical history.CBH was classified as absent, mild (<3 foci ≤2 mm), moderate (3-9 foci ≤2 mm or any focus ≤3-5 mm), or severe (≥10 foci ≤2 mm or any focus ≥6 mm).Clinical data were collected prospectively by trained clinical research nurses blinded to imaging.Infection was defined as culture positive sepsis; none had meningitis or were treated presumptively for meningitis; Two participants also had necrotizing enterocolitis defined as Bell's stage II or III (5).Cerebellar volume (mm 3 ) was determined as previously described (2).Three participants, including two with mild CBH and one with severe CBH, were excluded from volumetric analysis due to motion artifact.
Primary CGNP cultures.Mouse CGNPs were isolated as described (6) with the following modifications.Cerebella from P5-7 C57BL/6 male and female pups were digested in papain (Worthington) in DPBS for 30 minutes at 35°C.After papain inactivation with ovomucoid inhibitor (7) and trituration, the cell suspension was passed through a 40 µm nylon cell strainer (Corning Falcon) to eliminate large non-neuronal cells and obtain a single-cell suspension.To further remove glia, the suspension was incubated for 20 minutes on a poly-D-lysine (PDL, 100 µg/ml)-coated dish and repeated with a fresh dish to remove strongly adherent cells, yielding a final cell suspension of >95% granule neurons and progenitors (6).Cells were seeded onto PDL (500 µg/ml)-coated culture plates and maintained in a 5% CO2, 37°C incubator.Cell culture media was Neurobasal-A (Thermo Fisher Scientific), 1x B27 (Thermo Fisher Scientific), 2 mM GlutaMAX (Thermo Fisher Scientific), 20 mM KCl, and 1% penicillin-streptomycin (Thermo Fisher Scientific).Cells were treated with SHH (#464-SH, R&D Systems) alone or in the presence of fibrinogen (#341578, Millipore-Sigma) at concentrations indicated in the figure legends for up to 3 days in culture.For fibrin-coating experiments, PDL-coated tissue culture plates were incubated with a mixture of fibrinogen (1 μg/mL, #341578, Millipore-Sigma), thrombin (1 U/mL, Millipore-Sigma), and CaCl2 (8.7 mM, Millipore-Sigma) in 20 mM HEPES at 37ºC for 1 hour to form fibrin which was then dried onto the wells at 37ºC overnight.All conditions were tested in triplicate wells and repeated for N = 2 or 3 biological replicates as indicated in the figure legend.
Images were acquired with an Axioplan II epifluorescence microscope (Carl Zeiss) equipped with dry Plan-Neofluar objectives (10x 0.3 NA, 20x 0.5 NA, or 40x 0.75 NA), an Axiocam HRc CCD camera, and the Axiovision image analysis software; or the BIOREVO BZ-9000 inverted fluorescence microscope (Keyence) equipped with a Nikon CFI 60 Series infinite optical system and Keyence imaging software; All images were processed and analyzed in ImageJ.For animal studies, quantification on two or more nonadjacent sections per mouse was performed on either thresholded binary images or counting of cells by researchers blind to the treatment group.For in vitro studies, quantification of cells on 5-10 10x images per well was performed using ImageJ.
Immunoblots.Cells or tissue were lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with protease/phosphatase inhibitors (Millipore Sigma) and lysates cleared by centrifuging at 13,000xg for 15 minutes at 4ºC.Equal amounts of protein were loaded in 4%-12% bis-tris gels (Thermo Fisher Scientific) and analyzed by western blotting.Bands were visualized with HRPconjugated secondary antibodies (Cell Signaling Technology).Densitometry was performed using ImageJ with values for each band normalized to GAPDH loading controls from the same membrane.

Table S1 :
Baseline clinical and imaging characteristics of preterm infants by CBH *Both infants with NEC also had sepsis CBH, cerebellar hemorrhage NEC, necrotizing enterocolitis.CGA, corrected gestational age.WMI, white matter injury.IVH, intraventricular hemorrhage.

Table S2 :
Multivariate regression to assess associations with preterm infant cerebellar volume a Adjusted for CBH severity, sepsis, postnatal steroids, gestational age at birth, postnatal age at MRI, and total cerebral volume.CbVol, cerebellar volume.CBH, cerebellar hemorrhage.

Table S3 :
Mean body weight of experimental mouse groups randomized at postnatal day 2 (P2)